Summary The expression of protein-coding genes is a multi-step process that begins with transcription and RNA processing (capping, splicing and 3' end formation) in the nucleus followed by export of mature mRNA to the cytoplasm for translation. All of the steps in gene expression are coupled to one another via an extensive network of physical and functional interactions that remains to be understood. Defects in gene expression are a major cause of human diseases, and conversely, gene expression is one of the major cellular processes that can be harnessed to diagnose and treat human diseases. Thus, it is vital to achieve a detailed understanding of the gene expression pathway. The broad goal of this proposal is to identify the factors and determine the mechanisms for coupling among the transcription, splicing, and mRNA export machineries. The central goal of Specific Aim 1 is to determine how transcription is coupled to splicing. A current model proposes that early splicing factors (U1 snRNP and SR proteins) are co-transcriptionally recruited to pre-mRNA to initiate spliceosome assembly. The protein FUS, which associates with U1 snRNP, SR proteins, and RNA polymerase II, was identified as a strong candidate for coupling transcription to splicing. FUS also associates with the SMN complex, which functions in snRNP biogenesis. RNAi and biochemical work will be carried out to determine the roles of FUS in coupling transcription to splicing and in U1 snRNP biogenesis. In Specific Aim 2, a major effort is planned to determine how mutations in FUS cause the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). SMN-containing nuclear Gems are deficient in FUS knockdown HeLa cells and in ALS patient fibroblasts with a FUS mutation. Work is planned to determine whether Gem deficiency is a general phenotype in ALS. Splicing and snRNP biogenesis assays will also be carried out using nuclear extracts prepared from FUS knockdown HeLa cells and nuclear extracts containing FUS with ALS- causing patient mutations. These assays will also be performed using extracts prepared from motor neurons differentiated from mouse embryonic stem cells containing human ALS-causing patient mutations. This work is anticipated to add valuable insights into the pathogenesis of ALS as well as potentially leading to diagnostic tools for this disease. Finally, the central goal of Specific Aim 3 is to understand the function of the TREX complex, which is a highly conserved machinery that functions in mRNA export. TREX is linked to the spliceosomal Prp19 complex, and work is planned to determine how these complexes function in the export of both naturally intronless and spliced mRNAs. A combination of RNAi, factor recruitment assays, and RNA- protein interaction studies will be used to determine how these complexes function. Important insights into the essential cellular process of mRNA export is anticipated from these studies.